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ISRH-seq: a sensitive and robust methodology for R-loop profiling

ABSTRACT: R-loops serve as key modulators in various biological processes in humans and plants.Current high-throughput methodologies for R-loop profiling have their own intrinsic pros and cons. Therefore, any robust, sensitive, and amenable methods, complementary and/or advantageous to existing methodologies, are much needed for advancing R-loop biology. We here present an RNase H based cleavage combined with DNA polymerase-mediated in situ biotin-dATP/dCTP labeling of newly synthesized DNA for sequencing, termed ISRH-seq, an antibody-independent approach for strand-specific mapping of R-loops. After conducting a parallel comparison with DRIP-seq, we found that ISRH-seq can identify more R-loop peaks with the same sequencing depth. Moreover, ISRH- and DRIP-specific R-loops had distinct genomic and epigenetic features, exhibited distinct relationships with expression of overlapping genes, and functional divergence. Importantly, we found that ISRH-seq can be scalable down to 250-100ng DNA input, indicative of a robust and sensitive high-throughput methodology. In addition, it can be applied for R-loop profiling under stress conditions. Thus, our study provides a valuable orthogonal methodology for comprehensive profiling of R-loops in plants. It could be easily adapted to other non-plant systems, especially for these biologically important tissues or cell types with limited cell numbers, thereby advancing understanding of R-loop biology.

ORGANISM(S): Oryza sativa

PROVIDER: GSE232347 | GEO | 2024/05/16

REPOSITORIES: GEO

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Project description:Strand asymmetry in the distribution of guanines and cytosines, measured by GC skew, predisposes DNA sequences towards R-loop formation upon transcription. Previous work revealed that GC skew and R-loop formation associate with a core set of unmethylated CpG island (CGI) promoters in the human genome. Here, we show that GC skew can distinguish four classes of promoters, including three types of CGI promoters, each associated with unique epigenetic and gene ontology signatures. In particular, we identify a strong and a weak class of CGI promoters and show that these loci are enriched in distinct chromosomal territories reflecting the intrinsic strength of their protection against DNA methylation. Interestingly, we show that strong CGI promoters are depleted from the X chromosome while weak CGIs are enriched, a property consistent with the acquisition of DNA methylation during dosage compensation. Furthermore, we identify a third class of CGI promoters based on its unique GC skew profile and show that this gene set is enriched for Polycomb group targets. Lastly, we show that nearly 2,000 genes harbor GC skew at their 3’ ends and that these genes are preferentially located in gene-dense regions and tend to be closely arranged. Genomic profiling of R-loops accordingly showed that a large proportion of genes with terminal GC skew form R-loops at their 3’-ends, consistent with a role for these structures in permitting efficient transcription termination. Altogether, we show that GC skew and R-loop formation offer significant insights into the epigenetic regulation, genomic organization, and function of human genes. DRIP-seq was performed on genomic DNA extracted from human pluripotent Ntera2 cells. The DNA was either fragmented using HindIII, EcoRI, BsrGI, XbaI and SspI (DRIP-seq 1) or BamHI, NcoI, ApaLI, NheI and PvuII (DRIP-seq 2, two technical replicates). Input DNA was also fragmented with each restriction enzyme cocktail and sequenced alongside.

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ISRH-seq: a sensitive and robust methodology for R-loop profiling

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